Consumption of dietary carotenoids is associated with a decreased risk of cardiovascular disease and cancer. Humans obtain carotenoids in the diet by ingesting fruits and vegetables. Historically, it was thought that the most important role of some carotenoids was their metabolic conversion via cleavage at the central double bond to the essential micronutrient, vitamin A (retinol). There is intense recent interest in the "eccentric cleavage" of dietary carotenoids to apocarotenals and their possible oxidation to apocarotenoic acids. These metabolites of carotenoids may play important roles in the non-vitamin A activities of carotenoids. There are no quantitative analyses of the levels of these compounds in human foods and tissues. Demonstrations of the extent of metabolism of intact carotenoids to these compounds and studies of their mechanisms of action are lacking. We will address these gaps in knowledge. (1) We determine the substrate specificity and characterize the products of the two known human carotenoid-metabolizing enzymes (BCO1 &BCO2). These studies will also establish the relationship of cellular retinol-binding proteins and carotenoid cleavage enzymes in the metabolic function of these enzymes. (2) We will demonstrate the relevance of apocarotenoids in humans using analytical chemistry to characterize the presence and quantify the amounts of apo-carotenals and apocarotenoic acids in human plasma and tissues and in foods. This will be accomplished by quantitation of the immediate post- prandial plasma concentrations of parent carotenoids and metabolites after subjects consume a single serving of tomato juice containing nutritionally relevant amounts of beta-carotene or lycopene as well as by quantitation of steady state concentrations in plasma after 4 weeks of daily consumption. (3) We will use the LNCaP cell, a human prostate cancer cell that demonstrates lycopene-dependent growth inhibition and expresses BCO2 to look for apolycopenals and apolycopenoic acids in lycopene-treated cells to address whether and to what extent lycopene is metabolized in these cells. We will use the human monocyte/macrophage cell, U937, which is growth inhibited and induced to differentiate with carotenoid treatment, to look for metabolism of the parent carotenoids. (4) We will probe the biological (nutritional) function of carotenoid metabolites in multiple ways: (a) we will use cell lines that we have shown have characteristic functional responses to treatment with dietary carotenoids and ask whether these responses are obtained by direct treatment with the cleavage products and (b) we will ask whether these cleavage products and metabolites directly activate or antagonize ligand-dependent transcription factors. (c) We will feed the BCO1 knockout mouse both beta-carotene and lycopene to assess the extent of the metabolism of these carotenoids by BCO2 and the metabolic effects of carotenoid metabolites in the whole animal. The results will enhance our understanding of the molecular basis for the health-promoting effects of diets rich in fruits and vegetables. PUBLIC HEALTH RELEVANCE: Increased consumption of dietary carotenoids is associated with decreased risk of both cardiovascular disease and certain cancers. The proposed research will document the occurrence of new pathways of metabolism of dietary carotenoids (the health-promoting, colored pigments in fruits and vegetables) in humans. The research will also explore possible mechanisms that may explain the basis of the health-promoting effects of these components of the diet.